GB2037773A - Process for preparing stabilised vitamin D and compositions thereof - Google Patents

Abstract

A process for stabilizing vitamin D by the preparation of an inclusion complex of vitamin D with a cyclodextrin, which comprises forming a homogeneous solution from the vitamin D to be complexed and the cyclodextrin in aqueous ethanol at an elevated temperature, and separating the desired inclusion complex from the solution by cooling and/or by evaporation of solvent. The aqueous ethanol has an ethanol content (58% to 65% by volume) such that both vitamin D and the cyclodextrin are soluble. They can be formulated into pharmaceutical compositions, fodder premixes or fodders in conventional manner.

Description

SPECIFICATION Process for preparing stabilished vitamin D and compositions thereof The invention relates to a new method for the preparation of stable vitamin D - cyclodextrin inclusion complexes and pharmaceutical compositions containing them.

Rachitis, a metabolic disease most frequent in infancy, can be traced back to a vitamin D deficiency of the organism. The major consequences of this disease are disturbances in the bone development, damage and deformation in the spinal column, upper and lower limbs and the thorax, and softness of the vertex.

Moreover, without changes in calcium level, the phosphate level of the serum decreases.

Several compounds with vitamin D activity, capable of eliminating this syndrome, are known. Presently in human and veterinary therapy vitamin D3 (cholecalciferol, oleovitamin D3, activated 7-dehydrocholesterol) and dihydrotachisterol (DHT) have been introduced, whereas the utilization of other compounds with analogous effects, such as 25-hydroxycholecalciferol and 1 ,25-dihydroxycholecalciferol (hydroxylated derivatives of vitamin D3) and synthetic vitamin D analogues (such as 1-hydroxycholecalciferol and 5,6-trans-cholecalcifernl), is now only in the stage of clinical testing. All the above compounds can be adminstered orally as well. Their effects are stronger and faster, but shorter-acting, than that of vitamin D3.

Vitamin D3 is, however, an unstable compound. UV light converts itto suprasterol, and it loses its vitamin D activity as a consequence of ring closure.

Several problems emerge in connection with solid pharmaceutical compositions containing vitamin D3, because in these products the decomposition of vitamin D3 can hardly be controlled. The determination of vitamin Dg content is difficult, since one tablet contains 3000 international units (IU) of vitamin Dg on the average (one international unit is 0.025 ug), and this very small amount (75 1us) of the vitamin is dispersed in a large quantity of carrier. Under such conditions, because of the high liability of vitamin D3 to decomposition, the activity of the tablet is lost rather quickly.

Solid compositions are utilized not only in human therapy but also in animal husbandry. Fodder premixes with vitamin D3 content are widely used for veterinary purposes to prevent vitamin D3 deficiency in the animals. With such compositions the problem becomes even more severe. Fodder premixes, unlike compressed tablets and coated tablets used for humal therapy, in which the active agent is protected from oxygen to a certain degree, contain vitamin D3 in a relatively homogeneous distribution on a large surface. It is technically difficult to provide finely dispersed lipid-type substances, and the distribution on a large surface increases their liability to decomposition very much.

The essence of the methods suggested so far for stablizing vitamin D3 in solid pharmaceutical compositions is to use certain additives (antioxidants) in the preparation of tablets, or to coat the tablets with light and oxygen blocking layers. Although these substances increase the stability of the active agent owing to the inhibition of oxidation, they cannot effectively prevent the ring closure reaction, which is the primary factor in decomposition.

According to the method of the invention vitamin D3 and its derivatives with similar effects (e.g. the hydroxylated cholecalciferol derivatives mentioned above) are converted into cyclodextrin inclusion complexes. By this method, which is essentially an encapsulation in molecular dimensions, the conformation of the molecule is stabilized, and the possiblity of assuming a conformation required for ring closure is hindered by shielding the active groups with a cyclodextrin ring. Furthermore, as known, the use of cyclodextrin inclusion complex offers a protection against oxidation as well, since atmospheric oxygen is physically unable to attack the vitamin D3 molecule situated in the crystal lattice, presumably channel-type crystal lattice, of the cyclodextrin inclusion complex.

--Cyclodextrin - vitamin D3 inclusion complexes can be converted by known methods into conventional solid pharmaceutical compositions, such as tablets or coated tablets, with significantly improved stabilities.

Cyclodextrin inclusion complexes, besides stabilizing the vitamin D, have the additional significant advantage of being very fine, free-flowing powders easy to disperse, which can be distributed evenly in fodders by much simpler techniques than the lipid-type vitamin itself, which tends to be cohesive. According to a known method utilized already to overcome the problems of distribution, the lipid-type vitamins (such as vitamin D3) are microencapsulated, and the resulting microcapsules with more favourable dispersion characteristics are admixed with the fodder. The use of cyclodextrin inclusion complexes has the advantage that the complicated process of microencapsulation can be omitted.Furthermore the individual grains of the cyclodextrin inclusion complexes are smaller by about 2 to 3 orders to magnitude than those of the microcapsules, making the homogeneous distribution of the former even easier.

The preparation of vitamin D3 - cyclodextrin inclusion complex is described in the published Japanese patent application No. 76 128,417. According to this known process vitamin D3 is dissolved in an organic solvent, the solution is poured into a warm (50-60"C), stirred solution of p-cyclodextrin, the resulting mixture is cooled under stirring, whereupon the p-cyclodextrin inclusion complex of vitamin D3 separates. In this process only water-miscible organic solvents can be used to dissolve vitamin D3, since otherwise the solvent itself would form an inclusion complex. The disadvantage of this process is that the complexing of vitamin D3 is incomplete, since a part of vitamin D3 separates in micro-crystalline form upon admixing the solutions, thereby becoming unavailable for the complexing process.Consequently, the above process yields a heterogeneous product, also comprising unchanged cyclodextrin and microcrystalline vitamin D3 besides the required inclusion complex, and thus only a part of the starting amount of vitamin D3 can be stabilized by this known method.

Therefore, in order to increase the stability of the vitamin to the required extent, it is essential to complex all the starting vitamin D3.

The invention is based on the recognition that the above problems can be eliminated when the inclusion complex is separated from a homogeneous solution.

According to the invention, instead of admixing a solution of vitamin D with a cyclodextrin-containing solution, which involves the immediate precipitation of the complex and the microcrystalline vitamin from the liquid, the inclusion complex is formed by dissolving both components in one and the same solvent, and separating the product, containing vitamin D in complexed form alone, from this common homogeneous solution by crystallization at an appropriate rate.

More specifically our invention provides a process for the preparation of an inclusion complex of vitamin D with a cyclodextrin, which comprises forming a homogeneous solution from the vitamin D to be complexed and the cyclodextrin in aqueous ethanol at an elevated temperature, and separating the desired inclusion complex from the solution by cooling and/or by evaporation of solvent.

Several methods are known for the preparation of cyclodextrin inclusion complexes. An essential characteristic of these known methods is that complexing is performed in water or in an aqueous liquid containing a small amount of an organic solvent (almost exclusively an alcohol). It is known that the solubility of p-cyclodextrin passes a maximum as a function of ethanol concentration; thus e.g. at 37"C the maximum solubility occurs at an ethanol content of 30%. With increasing alcohol concentration the solubility of ss-cyclodextrin n decreases significantly, and the substance cannot be dissolved in dry alcohol.When studying the solubility characteristics of vitamin D3 we have observed that above an ethanol concentration of 60% vitamin D3 is fairly soluble in aqueous ethanol, and yields a solution which has technically acceptable concentration for complexing purposes. Moreover, below an ethanol content of 65%, the solubility of ss-cyclodextrin is still acceptable for complex formation. Thus it has been found, unexpectedly, that in a narrow range of ethanol concentration (i.e. from 58 or 60% to 65%) a common solution of technologically advantageous concentration can be formed from both components.

Complex formation may take place at an elevated temperature in the range 20 to 90"C, preferably about 60 or 50"C. Cooling can take place at a controlled rate to obtain a product of desired crystal size. The solvent is preferably evaporated by freeze drying.

The solubilities of vitamin D3 and p-cyclodextrin, measured at 50"C, are given in Table 1 as a function of the ethanol concentration.

TABLE 1 Concentration of ss-Cyclodextrin Vitamin D3 ethanol mmolii mmol/1 30% 85.4 40% 75.3 0.254 50% 66.0 2.54 60% 30.0 10.0 70% 9.53 26.6 80% 1.76 140.0 90% 360.0 According to the date of the table the two solubility curves intersect each other at an ethanol content of 64% (the solubility is expressed in mo 1/1); i.e. this ethanol concentration is the optimum for the formation of inclusion complexes.

A precondition of inclusion complex formation is that the foreign molecule, i.e. vitamin D3, should have appropriate dimensions, shape and polarity so that it can, either partially or completely, find room in the cavity of cyclodextrin and is able to form non-polar interactions or hydrogen bonds with cyclodextrin which favour the complex formation. From the dimensions of vitamin D3 it follows that one molecule of vitamin D3 is able to form an inclusion complex with two molecules of cyclodextrin. The two cyclodextrin molecules form an envelope or cage around the vitamin D3 molecule. Therefore, it is not recommended to perform the process below a 2:1 molar ratio of cyclodextrin to vitamin D3, and it is advantageous to use cyclodextrin in excess. Considering that an extremely low amount of vitamin D3 is required in both the pharmaceutical compositions for human therapy and the fodder premixes used in animal husbandry, the homogeneity of the distribution can be influenced favourably when the substance to be distributed - i.e. the cyclodextrin @@@@@@@@@ complex of vitamin D3 - is present in larger amounts. Therefore, it is preferred to use cyclodextrin and vitamin D3 in a molar ratio higher than 2:1, thus e.g. in a molar ratio of (3-6):1.

Vowing to their stability, the vitamin D3 - cyclodextrin inclusion complexes prepared according to the invention can be used to advantage in the preparation of pharmaceutical compositions and fodder premixes.

7 - prepare these compositions the inclusion complexes are mixed with conventional additives, such as -ai-riers, filling agents, flavouring agents, dyestuffs and/or formulation aids or excipients.

The pharmaceutical compositions can be presented in the form of tablets, capsules, powders, granulates, coated tablets, syrups, emulsions and suspensions. These compositions can be utilized both in human therapy and for veterinary purposes. Fodder premixes and fodders containing the complexes prepared according to the invention are compositions to be used specifically as veterinary substances.

The pharmaceutical and veterinary compositions may contain to advantage, beside the inclusion complexes prepared according to the invention, components providing calcium and phosphate ions as well.

The invention is illustrated in detail by the aid of the following non-limiting Examples.

Example 1 2000 ml of 60% aqueous ethanol are heated to 60 C, and 3.28 g of crystalline vitamin Dg are dissolved in it upder a continuous stream of nitrogen. When dissolution is complete, 60 g of ss-cyclodextrin (water content: 11.4%) are added, and this substance is dissolved under constant stirring and nitrogen introduction. When a completely clear solution is obtained, cooling is started under continuous stirring and nitrogen introduction, and the solution is cooled to room temperature within 5 hours at a uniform rate.The separated white, c istalline substance is filtered off and dried in air. 40 g of a substance with an average particle size of 20 to 9 < U, a humidity content of 7.8% and a vitamin D3 content of 7.6% are obtained; thus the yield is 91.4% calculated for vitamin Ds.

The formation of inclusion complex can be proved by X-ray powder diffraction measurements. The character;stic reflections (2 in degrees) of í3-cyclodextrin, vitamin D3 and of the inclusion complex are listed in Table 2.

TABLE 2 3-Cycljdextrin Vitamin D3 Inclusion complex 20 20 20 4 7 5.1" 6.7 9.0 6.8 11.7 10.60 8.9 17.8 1." 13.7" 15.3 15.8 q oO 18.0 19.6" The vitamin D content of the product is determined as follows: 20 mg of the product are dissolved in 1 ml of dimethyl formamide, the solution is diluted to 50 ml with 96% ethanol, and the extinction of the resulting mixture is measured at 264.5 nm.

Exan-le 2 One proceeds as described in Example 1 with the difference that the suspension obtained after crystalliration is evaporated in a rotary distillation apparatus at 30"C to one-third of the original volume, and the resulting thick crystal suspension is freeze-dried. The average grain size ofthe resulting product is 15 to 18 u, i.e. slightly smaller than that of the crystallized substance, and the yield is practically 100% for both ss-cyclodextrin and vitamin D. Freeze-drying has the advantage that a product even looser in structure is obtained, with a higher solubility in water than the crystallized substance.

Example 3 One proceeds essentially as described in Example 1 with the difference that 120 g of a so-called crude conversion product are used instead of 60 g of ss-cyclodextrin. The crude conversion product is prepared by contacting starch with cyclodextrin transglucosidase under the conditions used in the production of cyclodextrin, and subjecting the resulting substance, a mixture of cyclodextrins and partially decomposed starch, to spray-drying. Such crude conversion products contain generally 42 to 54% of ss-cyclodextrin. The inclusion complexes obtained by utilizing crude conversion product as complexing agent can be used to advantage primarily in the preparation of fodder premixes.

Example 4 One proceeds essentially as described in Example 1 with the difference that vitamin D3 is replaced by a same amount of 25-hydroxycholecalciferol. The yield and the quality of the product are practically the same as given in Example 1.

The stability of the product can be characterized by its resistance against heating, exposure to light, exposure to oxygen and exposure to metal salts.

The heat stability of the product was studied by thermogravimetric methods and by the UV spectrophotometric determination of vitamin D3 content.

The thermal analysis of the vitamin D3- ss-cyclodextrin inclusion complex was performed in the DSC cell of a Dupont 990 Thermal Analyser and in the micro-TG compartment of the same apparatus, under air stream.

DSC measurements were performed in aluminium crucibles, and TG measurements were performed in platinum crucibles. A sharp minimum appears in the DSC curve of ss-cyclodextrin below 1 OO"C, which corresponds to the elimination of the water content (about 14%). The next change (a sharp minimum) can be observed on the curve only above 290 C, which is due to the decomposition of ss-cyclodextrin during melting.

After the melting of the sample the decomposition becomes strongly exothermic, and under the conditions of the measurement the sample even flares up owing to the presence of atmospheric oxygen. The run of the DTG curve is therefore irregular between 290 and 365"C. According to the thermal analysis of vitamin D3 the substance melts at about 85"C, and then the melt is oxidized, as proved by a slight weight increase and the exothermic nature of the process (the decomposition of vitamin D3 starts at about 1700C and proceeds in two steps).Decomposition is completed at about 550"C. For a physical mixture of 93% of ss-cyclodextrin and 7% of vitamin D3 the thermoanalytical curve is essentially the same as that of pure B-cyclodextrin, with the only difference that a weight loss appears first at about 170 , due presumably to the decomposition of vitamin D3.

This mixture, like pure ss-cyclodextrin, also flares up at about 300"C.

The DTG curve of vitamin D3 - P-cyclodextrin inclusion complex shows a characteristic minimum at 255 C, and the DSC curve also has a decrease in the same region. The next minimum appears in the curve at 290 C.

Above this temperature the substance, instead of flaring up, burns slowly with cracking between 400 and 500 C. By comparison with data for other cyclodextrin inclusion complexes, this behaviour proves unambiguously that the substance prepared according to the invention is really an inclusion complex of vitamin D3.

In the next test the heat stability of the inclusion complex prepared according to Example 1 (Sample 3) was compared with that of a product prepared as described in the published Japanese patent application No. 76 128,417 (Sample 2) and with that of the physical mixture of 6.7% of vitamin D3 and 92.3% of ss-cyclodextrin (Sample 1).

Layers of the substances under examination, 1 mm in thickness, were subjected to heat treatment in air at 80"C. The heat treated substances were sampled at regular intervals, and the vitamin D3 content was determined photometrically after dissolving the samples in small amounts of dimethyl formamide and diluting the solution with 96% ethanol. The results are given in Table 3.

TABLE 3 Time Sample 1 Sample 2 Sample 3 (days) (D3 = 7.1%) (D3 = 7.6%) 2 no vitamin D3 80.2 94.7 can be 5 detected 74.9 88.9 even after 8 the first 68.5 81.0 24 hours 10 63.0 76.0 13 60.4 72.3 17 51.3 67.3 20 42.0 64.1 24 43.7 57.9 28 31.5 54.8 43 25.7 49.1 The data of Table 3 show that even after 24 hours of heating no vitamin D3 can be detected in the physcial mixture of vitamin D3 and B-cyclodextrin, used as reference substance. After 43 days of heating the sample prepared according to the invention relates 49.1% of its vitamin D3 content, whereas the sample prepared according to the cited reference only 25.7%.

The solubility of vitamin D3 in the form of ss-cyclodextrin inclusion complex increases characteristically, as indicated by the following series of tests: 50 g of crystalline vitamin D3, 200 mg of crystalline vitamin D3 - P-cyclodextrin inclusion complex prepared as described in Example 1, or 200 mg of freeze-dried vitamin D3 - ss-cyclodextrin inclusion complex prepared as described in Example 2, respectively, were introduced into 100 ml of a Britton-Robinson buffer solution (pH = 7) at 37"C under vibrational stirring. The resulting mixtures were stirred intensely, the mixtures were samples at regular intervals, the samples were filtered immediately, the filtrates were diluted with ethanol, and the extinctions of the resulting solutions were measured at 265 nm.The amount of dissolved vitamin D3 was calculated from the extinctions measured. The results are summarized in Table 4.

TABLE 4 Dissolved vitamin D3, mg/100 ml Time Vitamin D3 Product of Product of (Minutes) Example 1 Example 2 5 0 0.210 0.232 10 0 0.180 0.252 20 0 0.192 0.270 30 0 0.220 0.234 40 0 0.204 0.336 50 0 0.214 0.238 60 0 0.202 0.312 75 0 0.212 0.336 90 0 0.220 0.340 Although a very slight increase in extinction was observed with crystalline vitamin D3, this was due not to the dissolution of the substance, but, as verified spectroscopically, to the appearance of an unidentified decomposition product. Thus crystalline vitamin D3 was observed to be insoluble. On the other hand, the crystalline and freeze-dried vitamin D3 - ss-cyclodextrin complexes yield solutions containing 0.220 mg/100 ml and 0.340 mg/100 ml of vitamin D3/ indicating a significant increase insolubility, which is very important with respect to in vivo resorption.

The stability of vitamin D3 - ss-cyclodextrin inclusion complex against the action of oxygen was examined in a Warburg apparatus at 37"C under pure oxygen atmosphere: The following samples were tested: Sample 1: pure vitamin D31 Sample 2: a product prepared according to the published Japanese patent application No.76 128,417, Sample 3: the product of Example 1.

The oxygen uptake related to 1 mg of vitamin D3 as a function of time is given in Table 5.

TABLE 5 Time Oxygen uptake (ul of oxygen/mg of vitamin D3) (hours) Sample 1 Sample 2 Sample 3 50 6 17 6 100 14 31 9 150 48 ' 38 12 200 83 44 14 250 125 50 16 300 132 52 16 350 140 55 16 It appears from the table that vitamin D3, after an initial induction period, takes up oxygen according to an S-shaped curve, a-nd ultimately one milligram of it consumes about 140 ul of oxygen. The oxygen uptake of the crystalline vitamin D3 - p-cyclodextrin inclusion complex prepared according to the published Japanese patent application No. 76 128,417 is 39.5% of the above value, whereas the complex prepared according to Example 1 takes up only 1 1.20,/0 of the oxygen consumed by pure vitamin D3.

Light stability was examined as follows: A physical mixture of 6.7No of vitamin D3 and 92.3% of í,-cyclodextrin (Sample 1), a vitamin D3 ss-cyclodextrin inclusion complex with a vitamin D3 content of 7.1%, prepared as described in the published Japanese patent application No. 128,417 (Sample 2), and a complex prepared according to Example 1 with a vitamin D3 content of 6.9% (Sample 3) were tested. The samples were spread onto a Petri dish in a layer of i mm thickness, and were exposed to light (wave-length: 400 to 600 nm, illumination power: 2900 lux) for 320 hours.Samples were taken from the illuminated substances at regular intervals, the samples were dissolved in small amounts of dimethyl formamide, the solutions were diluted with 96 o ethanol, and vitamin D3 content was determined by UV spectrophotometry. The results of these measurements are given in Table 6.

TABLE 6 Time Vitamin D3 content related to the starting value, % (hours) Sample 1 Sample 2 Sample 3 0 100.0 100.0 100.0 32 80.6 101.0 101.6 48 82.1 97.2 101.6 64 76.0 92.1 96.7 96 52.2 88.7 98.3 112 58.3 87.1 95.1 160 47.1 87.1 95.1 320 45.5 82.6 95.1 The data of Table 6 indicate that the substance prepared according to the invention is the most stable under the above conditions.

The stability in the presence of minerals was examined as follows: The effect of copper salts was examined on the decomposition of vitamin D3 when admixed or complexed with 13-cyclodextrin. In the test, a physical mixture of 93% of p-cyclodextrin and 7% of vitamin D3 (Sample 1) and the complex prepared according to Example 1 (Sample 2) were investigated. The samples were admixed with finely powdered crystalline copper sulfate in a 1:1 weight ratio, and the mixture was thoroughly blended and homogenized in a mortar. The samples were exposed to air at room temperature, and the vitamin D3 content was determined monthly. The results are given in Table 7.

TABLE 7 Time Vitamin D3 content related to the starting value, % (months) Sample 1 + CuS04 Sample 2 + CuS04 0 100.0 100.0 1 61.2 90.6 2 45.2 83.5 3 38.7 82.1 4 29.2 78.6 5 22.1 77.2 The data of Table 7 clearly indicates that complexing stabilizes vitamin D3 to a great extent against the effect of copper salts which catalyze decomposition.

The biological activities of vitamin D3 - ss-cyclodextrin inclusion complexes were studied as follows: Three tests were carried out to prove that (i) the activity of the inclusion complex exerted on rats suffering from rachitis is the same or even better than that of vitamin D3 used in the same dosage (Test 1); (ii) vitamin D3 is resorbed more efficiently from the inclusion complex than from pure vitamin D3 (Test 2); and (iii) heat treated samples of vitamin Dg inclusion complexes retain their biological activities, whereas a considerable loss of biological activity occurs with heat treated free vitamin D3 (Test 3).

Test I: Female rats weighing 50 g in average were kept for 36 days on a rachitogenic diet of the following composition: wheat fiour 33% maize flour 33% wheat gluten 15% gelatine 15% CaCO3 3% NaCI 1% At the end of this period a substantial lag in weight gain, as compared to the animals kept on normal rat diet, could be observed (see the data of Table 8), and significant changes appeared in the phosphate and calcium levels of the serum.

In the first test series the rachitic rats, kept for 36 days on a rachitogenic diet, were treated with 5 lU/day of vitamin D3 for 2 days and then with 101U!day of vitamin D3 for further 23 days. The members of the first group received vitamin D3 in the form of a standard composition, whereas those of the second were treated with the vitamin D3 - ss-cyclodextrin inclusion complex prepared according to Example 1. The following results were obtained: Body weight: The increase in body weight was almost the same for the two groups during the first 2 weeks, but later on the animals treated with the inclusion complex showed a greater weight gain.

Phosphate and calcium level of the serum: For the animals treated with standard vitamin D3 the inorganic phosphate level of the serum did not reach that of the controls during the total 25 days' observation period; on the 18th day the phosphate level was still only 75% of that of the controls. On the contrary, the serum phosphate level of the animals treated with the inclusion complex reached the normal value within one week. Measurements of inorganic phosphates incorporated by the bones also indicate that phosphate incorporation is faster upon the introduction of the inclusion complex.

The serum calcium level of the animals treated with standard vitamin D3 reached only about 90% of that of the controls by the end of the test period; whereas in the group treated with the inclusion complex, the serum calcium level was normalized one week after starting the treatment.

Morphology of the bones: After keeping the animals on a rachitogenic diet for 50 days there is a clearly visible knob-like thicknening of tibia epiphysis, the ossification zone is extended and chondrous, and the anterior and posterior surfaces of the epiphysis consist of strongly extending cartilage. Bones of such character can be graded as class 0.5 of the Bills scale ("very severe deformation"). On animals treated with standard vitamin D3 no change can be observed by the 7th day of the treatment, whereas the animals treated with the inclusion complex start to recover, and the status of the bones corresponds to class 1.75-2.0 of the Bills scale ("moderately severe"). At this stage the bifuration of the cartilage sets in, and the chondrous zone starts to retract.Only a minimum recovery can be observed on the animals treated with standard vitamin D3 after a 22 days' treatment, whereas in the animals treated with the cyclodextrin inclusion complex the posterior knob of the tibia I epiphysis disappears, and the chondrous zone narrows significantly. On the 25th day of treatment the chondrous zone is narrow already in the animals treated with vitamin D3, the upper bone trabecule is, however, not normal yet. In the animals treated with the cyclodextrin inclusion complex a relatively well developed trabecule appears by this time. The animals of both groups are already in the state of "slight rachitis"; those treated with vitamin D3 can be graded as class 2.5, whereas those treated with the complex as class 3.0 of the Bills scale.Animals treated with the inclusion complex are about two weeks ahead of those treated with vitamin D3 with respect to the recovery of the rachitic state.

Test 2: In this test hypervitaminosis is provoked in the animals to demonstrate that vitamin D3 is resorbed more efficiently from the inclusion complex than from the non-complexed substance. According to the literature the dosage of vitamin D3 inducing permanent nephrocalcinosis (i.e. the toxid level) is 200 IU. Rats kept on a rachitogenic diet for 36 days were treated with 100 IU of vitamin D3 in free or complexed state, respectively.

Upon this treatment the movement of the animals became dynamic, and their coarse hair normalized. The body weight of the animals rapidly approached that of the controls (see Table 8).

After 4 days of the treatment all the animals treated with vitamin D3 - cyclodextrin inclusion complex perished. The autopsy of the animals showed the occurrence of high bile secretion, inflated intestines, and the cortical substance of the kidneys was light in colour characteristic of haemoglobin-urea, which was also demonstrated by a positive benzidine reaction. This test was repeated with non-rachitic intact rats weighing 150 g to arrive at the same results. Thus the vitamin D3 - {S-cyclodextrin inclusion complex, when administered in a daily dosage of 100 IU, always induces hypervitaminosis, whereas this could not be observed with free vitamin D3 administered in the same dosage.This indicates that vitamin D3 is resorbed more efficiently from the inclusion complex than from the non-complexed substance.

Test 3: In this test the biological activities of standard vitamin D3, standard vitamin D3 stored at 60"C for 48 hours, and vitamin D3 - ss-cyclodextrin inclusion complex stored at 60"C for 48 hours were compared.

Female rats weighing 50 g, kept previously on a rachitogenic diet for 36 days, were treated with vitamin D3 corresponding to a nominal dosage (i.e. the vitamin D3 content measured priorto heat treatment) of 100 lU/day.

The highest weight gain could be obtained with the heat-treated vitamin D3 - -cyclodextrin inclusion complex; the body weight of the animals closely approached that of the controls even on the 12th day. A smaller but still significant weight gain could be observed on the animals treated with standard vitamin D3, whereas the animals receiving heat-treated vitamin D3 showed significantly lower weight gain.

In rats maintained on a rachitogenic diet for 36 days the serum phosphate level decreases by more than one order of magnitude in comparison with the controls. When the heat-treated inclusion complex is administered to these animals, the serum phosphate level reaches or even slightly exceeds that of the controls even after 3 days. This indicates that, despite of heat treatment, the vitamin stabilized in the form of inclusion complex retains its activity. The efficiency of heat-treated standard vitamin D3 is much lower than that of the heat-treated complex. As shown by the examination of serum alkaline phosphatase and serum calcium levels, the activity of the heat-treated inclusion complex is hig her than that of the standard vitamin D3, and much higher than that of the heat-treated standard substance.

TABLE 8 Weight gain of rats kept on rachitogenic diet in comparison with the controls, given as a function of time Time Body weight of the animals, g (days) Vitamin D3 Heat-treated Heat-treated Controls vitamin D3 complex 1 51 51 51 51 12 57 50 55 80 24 60 61 61 103 36 69 68 71 116 start of vitamin treatment 42 79 83 95 136 48 140 105 198 203

Claims (19)

1. A process for the preparation of an inclusion complex of vitamin D with a cyclodextrin, which comprises forming a homogeneous solution from the vitamin D to be complexed and the cyclodextrin in aqueous ethanol at an elevated temperature, and seperating the desired inclusion complex from the solution by cooling andor by evaporation of solvent.

2. A process as claimed in claim 1, wherein said vitamin D comprises vitamin D3.

3. A process as claimed in claim 1, wherein said vitamin D comprises 25-hydroxycholecalciferol.

4. A process as claimed in any of the preceding claims, wherein said cyclodextrin is 1-cyclodextrin.

5. A process as claimed in claim 1, wherein an inclusion complex of vitamin D3 with íA-cyclodextrin is formed.

6. A process as claimed in any of the preceding claims, wherein one mole of said vitamin D is reacted with 2 to 6 moles of cyclodextrin.

7. A process as claimed in claim 6 wherein as least 3 moles of cyclodextrin are employed per mole of vitamin D.

8. A process as claimed in any of the preceding claims, wherein said aqueous ethanol has an ethanol content of 58 to 65% by volume.

9. A process as claimed in claim 8 wherein said aqueous ethanol has an ethanol content of about 60% by volume.

10. A process as claimed in any of the preceding claims, wherein said elevated temperature is in the range 20 to 90"C.

11. A process as claimed in claim 10 wherein said elevated temperature is about 60or.

12. A process as claimed in any of the preceding claims, wherein the desired inclusion complex is separated by cooling the reaction mixture.

13. A process as claimed in claim 12 wherein said cooling is performed at a controlled rate to obtain a complex of desired crystal size.

14. A process as claimed in any of claims 1 to 11, wherein the desired inclusion complex is separated by freeze-drying the reaction mixture.

15. A process as claimed in claim 1, substantially as illustrated in any one of Examples 1 to 4.

16. A vitamin D-cyclodextrin complex made by the process of any of claims 1 to 15.

17. A pharmaceutical composition containing an effective amount of the complex of claim 16 together with a pharmaceutically acceptable carrier or excipient.

18. A fodder premix containing an effective amount of the complex of claim 16 together with conventional fodder premix components.

19. Afodder having the complex of claim 16 uniformly distributed therein.